1. Field of the Invention
The present invention relates in general to the fields of optical detection methods, immunology, and cellular biology, and, more particularly, to methods of assessing the state of the immune system, and, even more particularly, to methods of measuring CD4+ T cells, as typically carried out in HIV-infected patients.
2. Description of Related Art
Obtaining accurate and reliable measures of CD4+ T lymphocytes (CD4+ T cells) is essential to assessing the immune system and managing the health care of persons infected with human immunodeficiency virus (HIV). The pathogenesis of acquired immunodeficiency syndrome (AIDS) is largely attributable to the decrease in CD4+ T cells, and progressive depletion of CD4+ T cells is indicative of an unfavorable prognosis.
In HIV-infected adults and adolescents, measurement of the number of CD4+ T cells per volume of blood (referred to as the CD4+ T cell count or simply the CD4+ count, with the understanding that the measured quantity is a concentration) is used as a measure of disease progression and to establish decision points for initiating and monitoring antiretroviral therapy. The CD4+ T cell count in infected patients decreases as HIV infection progresses, and patients with lower CD4+ T cell counts have a poorer prognosis than patients with higher counts.
A wide variety of technologies have been used for measuring CD4+ T cells. The most widely used assays for measuring CD4+ T cells are based on flow cytometry. Cells of interest, e.g., CD4+ T cells, in a sample are labeled with fluorescently labeled antibodies that bind specifically to cellular antigens whose joint expression identifies the cells of interest. The cells are passed in a fluid stream essentially one at a time through a detection region in which any fluorescent labels bound to a cell are optically detected. Cells of interest are identified by the joint detection of fluorescent labels bound to the cellular antigens whose joint expression identifies the cells of interest, and counted. Reagents and flow cytometers suitable for measuring CD4+ T cells are commercially available from, for example, BD Biosciences (San Jose, Calif.).
Methods of counting the number of CD4+ T cells in a sample using fluorescence microscopy have been described. Magnetic separation has been used to move the cells into position prior to analysis. For example, Tibbe et al., 2001, Cytometry 43:31-37, describe methods in which the CD4+ T cells are magnetically separated into a detection region at the top of a sample chamber, the region is optically scanned to obtain an image of the region, individual cells are identified using image processing algorithms, and the number of CD4+ T cells in the scanned region are counted. The requirements of scanning the sample add to the complexity and cost of the instrument. Assays based on identifying and counting cells of interest depend on the ability of the image processing to accurately identify the individual cells, and are sensitive to the density of the cells in the region.
Magnetic immunoassays have been described in which analyte-specific antibodies conjugated to magnetic particles are used to magnetically label a target analyte to facilitate magnetic separation of the analyte from the sample solution. Typically, after the magnetically labeled analyte has been concentrated against the side or bottom of the sample chamber, the sample fluid is removed. Such assays require sample handling fluidics to separate the captured analyte from the sample fluid and are inherently multi-step.
U.S. Pat. No. 5,945,281 describes a magnetic immunoassay in which a labeled target analyte is magnetically separated from a sample fluid and moved from a sample chamber into a detection region for optical analysis. The sample is added to sample chamber containing a magnetic capture reagents and a label such that target analyte in the sample forms a complex with the magnetic capture agent and the label. An electrical potential is applied to the complex to transport the complex to a detection region, and the presence of the complex in the detection region is determined.
U.S. Pat. Nos. 6,858,440; 6,645,777; 6,630,355; and 6,254,830; each incorporated herein by reference, describe a magnetic focusing immunosensor for magnetically concentrating pathogenic bacteria in a food sample onto the side of a sample container and optically detecting the concentrated cells through the side of the sample container. The magnetic focusing immunosensor comprises a focusing magnet and fiber optics attached to the side of the magnet for transmitting excitation and detection light.